Bacterial culture
Bacteroides thetaiotaomicron DS1880, which was isolated from human feces, was cultivated in tryptic soy broth (BD, Sparks, MD, USA) with 5% horse blood under anaerobic conditions at 37 ℃ for 36 h. The bacterial culture was incubated at 65℃ for 30 min and centrifuged at 3,000 g for 10 min. The supernatant was collected in a new tube and kept at -70℃ until use.
Cell Culture And Reagents
The human CRC cell lines HCT116 and LS174T were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 at 37˚C. Sodium propionate (SP; P5436) was purchased from Sigma-Aldrich. MG132 (M7449) and cycloheximide (CHX; C4859) were purchased from Sigma-Aldrich. BIX01294 (ab141407) was purchased from Abcam.
Cell Viability Assay
Cell Counting Kit-8 (CCK8; Dojindo Laboratories) was used to conduct the cell viability assays. Cells were seeded in 6-well plates at 4 × 105 cells/well and incubated for 24 h. After BT sup treatment, SP treatment or siRNA transfection for 48 h or BIX01294 treatment for 24 h, CCK8 solution and RPMI-1640 medium with 10% FBS were added to each well and incubated with 5% CO2 at 37˚C for 2 min or 5 min. The absorbance was assessed using a microplate reader at 450 nm. For crystal violet staining, the cells were fixed with methanol for 5 min and stained with 0.1% crystal violet after BT sup treatment, SP treatment, siRNA infection for 48 h or BIX treatment for 24 h.
Fluorescence-activated Cell Sorting (facs) Analysis
After treatment with BT sup, SP, knockdown of EHMT2 for 48 h or BIX for 24 h, the cells were collected and incubated with the Muse Annexin V and Dead Cell Assay kit (MCH100105; Merck) for 20 min at room temperature. For analysis with the Muse™ Caspase-3/7 Kit (MCH100108; Merck), the cells were incubated with caspase 3/7 reagent (Merck) for 30 min in a humidified atmosphere with 5% CO2 at 37˚C. After incubation, the cells were incubated with Caspase 7-AAD (Merck) for 5 min at room temperature. After incubation, ~ 5 × 104 cells were analyzed using a Muse Cell analyzer (Merck). The FACS results were analyzed using Muse 1.6 Analysis software (Merck).
Sirna Transfection
siRNA duplexes against EHMT2 (siEHMT2; 5'-GCAAAUAUUUCACCUGCCATT-3', 5'- UGGCAGGUGAAAUAUUUGCTT-3'), TNFAIP1 (siTNFAIP1; 5'- CUCUCAGGUCAGG UACCUUTT-3', 5'-AAGGUACCUGACCUGAGAGTT-3'), and HECTD2 (siHECTD2; 5'-CAUUGAAGACUCUGGGAUUTT-3', 5'-AAUCCCAGAGUCUUCAAUGTT-3') were purchased from Bioneer Co., Ltd. Negative control siRNA (siCont; 5'-AUGAACGUGAAU UGCUCAATT-3', 5'-UUGAGCAAUUCACGUUCACTT-3') was used as a control. The siRNAs (100 nM) were transfected into cancer cell lines using RNAiMax (Invitrogen) for 48 h [29].
Semiquantitative Reverse Transcription Pcr And Quantitative Real-time Pcr
Total RNA was isolated from the indicated cell lines using a Qiagen RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA aliquots of 1 µg were then reverse-transcribed using the iScript™ cDNA synthesis kit (Bio-Rad) according to the standard protocols provided by the manufacturer. For semiquantitative RT-PCR, cDNA was used as a template for PCR using AccuPower® HotStart PCR PreMix (Bioneer). Quantitative RT-PCR (EHMT2: annealing temperature 55˚C, 35 cycles; ACTB: annealing temperature 58˚C, 28 cycles) was performed using the SimpliAmp Thermal Cycler (Applied Biosystems) following the manufacturer's instructions. Quantitative real-time PCR was performed on cDNA samples using Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix, and the signal was detected using the AriaMx Real-Time PCR System (both from Agilent Technologies). The fluorescence threshold value was calculated using Agilent Aria 1.6 software (Agilent Technologies) (20,21). The PCR primers were as follows: EHMT2 forward, 5'-GGGCTACTGCCTCTTCTACG AGTC-3' and reverse, 5'-GTCTTTGTACTGCCGGTCCT CGTAG-3'; TNFAIP1 forward, 5'- CATCACATCCCTAAAGGAGGAGG-3' and reverse, 5'-AGCAGGTGGTCGTCAGAGTT-3'; HECTD2 forward, 5'-ATTCGTTTCCCTCCCTGCTG-3' and reverse, 5'-AGTTCACC TGCACATCTTGTTT-3'; and ACTB forward, 5'-ACTCTTCCAGCCTTCCTTCC-3' and reverse, 5'-CAATGCCAGGGTACATGGTG-3'.
Western Blot Analysis
The cells were washed once with phosphate-buffered saline (PBS) and then lysed in cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and 1X protease inhibitor cocktail). Cell lysates were centrifuged at 14,000 x g for 20 min at 4˚C and then boiled in 5X sample buffer following protein determination (BSA, 23208; Thermo Fisher Scientific). The protein samples were subjected to western blot analysis. For western blot analysis, nitrocellulose membranes (1620145; Bio-Rad ), blocking reagent (5% skim milk, 1 h at room temperature), and 4–20% precast gels (456–1094; Bio-Rad) were used with the indicated antibodies at a 1:1,000 dilution ratio (22). The samples were stained with the following antibodies: EHMT2 (ab185050) and TNFAIP1 (ab86934) from Abcam, PARP (9542) from Cell Signaling Technology, FLAG (F1804) from Sigma-Aldrich, and HA (sc-805) and ACTB (sc-47778) from Santa Cruz Biotechnology. Secondary antibodies (rabbit; sc-2357, mouse; sc-2031; Santa Cruz Biotechnology) were incubated at room temperature for 1 h, and ECL solution (170–5060; Bio-Rad) was used for visualization. A chemiluminescence imaging system (Mini HD9; UVitec) was used for imaging.
Immunoprecipitation
pCAGGS-n3FC (Mock), pCAGGS-n3FCEHMT2 (3 × FLAG-EHMT2), and Ub-HA were transfected into HCT116 cell lines using FuGENE® 6 Transfection Reagent (Promega) for 48 h. Transfected HCT116 cells were lysed with cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and 1X protease inhibitor cocktail). In a typical immunoprecipitation reaction, 800 µg of whole-cell extract was incubated with an optimum concentration of primary antibody and ANTI-FLAG® M2 Affinity Gel (A2220; Sigma-Aldrich). After the beads had been washed three times in 1 ml of Tris-buffered saline (pH 7.6), proteins that bound to the beads were eluted by boiling in 2X sample buffer (Thermo Scientific).
Chromatin Immunoprecipitation
ChIP was performed with Magna ChIP A/G (Magna0013 and Magna0014; Millipore) following the manufacturers’ instructions. HCT116 cells were transfected with siCont and siEHMT2 for 48 h or treated DMSO and BIX for 24 h crosslinked with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature, and quenched with 1X glycine (Millipore) for 5 min at room temperature. Then, the HCT116 cells were washed with cold 1X PBS (containing 1X Protease inhibitor Cocktail Ⅱ). After nuclear extraction, the chromatin solution was sonicated using a Bioruptor® Pico sonication device (B01060010; Diagenode) with 15 cycles of 30 seconds ON and 30 seconds OFF to obtain 200–1000 bp chromatin fragments. Sheared chromatin was incubated with 2 µg of H3K9me2 (ab1220; Abcam) antibody or 2 µg of H3K9ac (ab4441; Abcam) antibody with 20 µl of Magna ChIP A/G magnetic beads (Millipore) overnight at 4 °C. The complexes were incubated with ChIP elution buffer and RNase A mixture for 30 min at 37 °C and then incubated with proteinase K for 2 h at 62 °C. After DNA purification using spin columns, the samples were analyzed by semiquantitative PCR using TNFAIP1 Primer. The primers were as follows: TNFAIP1 P1 forward 5’-CTGGCAGCCGAACACAAGT-3’, and reverse, 5’-CCAA GCCAGATTCATGGGAGT-3’ ,TNFAIP1 P2 forward 5’-ACTCCCATGAATCTGGCTTGG-3’, and reverse ,5’-GCTCAGATGCTCAGACACGC-3’.
Immunocytochemistry
Cultured cells were fixed in 4% paraformaldehyde at room temperature for 10 min, permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 10 min and blocked with 5% bovine serum albumin in PBS for 30 min. Fixed cells were incubated with anti-EHMT2 antibody (ab185050; Abcam) and anti-TNFAIP1 antibody (ab86934; Abcam) overnight at 4 °C and stained with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Fluorescent images were obtained using a CELENA® S Digital Imaging System (Logos Biosystems).
Immunohistochemistry
Paraffin-embedded sections of colon tumor tissue array (T8235722-2, Biochain) were processed in a microwave (90℃) with antigen-retrieval solution (pH 9) (S2367; Dako), treated with a peroxidase-blocking reagent, and then treated with a protein-blocking reagent (K130, X0909; Dako). Tissue sections were incubated with rabbit anti-EHMT2 antibody (68851; CST) and rabbit anti-TNFAIP1 antibody (ab86934; abcam) followed by incubation with an HRP-conjugated secondary antibody (Dako). Immunoreactivitywas visualized with a chromogenic substrate (Liquid DAB Chromogen; Dako). Finally, tissue specimens were stained with Mayer's hematoxylin solution (Hematoxylin QS; Vector Laboratories) for 5 seconds to discriminate the nucleus from the cytoplasm.
Rna-seq And Analysis
Using TruSeq RNA Sample Preparation Kit V2, purification and library construction were carried out with total RNA, and Illumina HiSeq 2500 machines (Illumina, San Diego, CA, USA) were used for sequencing with a read length of 2 × 100 bases. FastQC v.0.11.4 was used for the quality of the paired-end reads. Cutadapt v.1.15 and Sickle v. 1.33 was used for filtering low-quality reads and adaptors. Cufflinks version 2.2.1 was used for calculation of FPKM (fragments per kilobase of transcripts per million mapped reads) values. Cuffdiff was used to select differentially expressed genes (DEGs) (fold change > 2). All Gene Ontology and KEGG pathway enrichment analyses were performed with DAVID ver. 6.8 and CluGO ver. 2.5.5 in Cytoscape ver. 3.7.1. GSEA analysis was performed with GSEA ver. 4.0.1.
Mouse Experiment
To establish a xenograft mouse model, HCT116 cells were implanted into the flanks of six-week-old Balb/c-nu female mice (Orient Bio. Sungnama, Korea). BIX01294 was intraperitoneally injected into mice every three times a week. Tumor size was measured twice every week, and the tumor volume was calculated by length (L) × width (W) × height (H). On day 23, all mice were sacrificed. The animal experiments were approved by the Committee on Animal Experimentation of the Korea Research Institute of Bioscience and Biotechnology.
Statistical analysis
The results are expressed as the mean ± SD (error bars) of three independent experiments. One-way analysis of variance (ANOVA) with the Bonferroni post hoc test was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The cut-off for significance was p < 0.05.